Peroxynitrite is a powerful oxidant and is suspected of being harmful to living cells. Under physiological pH, it can nitrate the tyrosine residues in proteins. To investigate the nature of this nitration and the effect of this irreversible modification on the important reversible tyrosine-phosphorylation regulatory mechanism, we carried out studies using synthetic peptides patented after the tyrosine phosphorylation site of p34cdc2, which is a protein kinase that plays a major role in the regulation of cell cycles. The amino acid sequence of the synthetic peptide, termed cdc2(6-20) is KVEKIGEGTYGVVYK, which is a good substrate for lck kinase p56lck, a member of the src-family of tyrosine kinases. Our results revealed (1) peroxynitrite can nitrate both Try-15 and 19 of cdc2(6-20)NH2 and (2) when Tyr-15, or both Tyr-15 and 19, in cdc2(6-20)NH2 were replaced by nitrotyrosine, both synthetic peptides failed to be phosphorylated by lck kinase. However, when only Tyr-19 is replaced by nitrotyrosine, the rate of phosphorylation is reduced by 20%. These data confirm that Try-15 is the phosphorylation site and nitrated tyrosine cannot be phosphorylated. In addition, nitration of a non-phosphorylation site tyrosine can exert an inhibitory effect on the phosphorylation of the non-modified tyrosine.